猫杯状病毒HRB-SS株感染性cDNA克隆的构建与病毒的拯救  

Construction of an infectious cDNA clone of FCV HRB-SS strain and virus rescue

作　　者：孙伟尧 宋海军 刘春国[1] 杨德成[1] 王靖飞[1] SUN Wei-yao;SONG Hai-jun;LIU Chun-Guo;YANG De-cheng;WANG Jing-fei(State Key Laboratory for Animal Disease Control and Prevention/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Zhangjiakou Center for Animal Disease Control and Prevention,Zhangjiakou 075000,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069 [2]张家口市动物疫病预防控制中心,河北张家口075000

出　　处：《中国预防兽医学报》2023年第10期1080-1084,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室自主研究课题(SKLVBP202121)。

摘　　要：为构建猫杯状病毒(FCV)HRB-SS株的感染性克隆,本研究合成3’端含有丁肝病毒核酶(HdvRz)序列的HRB-SS株全长基因组序列,将该序列插入p OK12-CMV载体,获得含FCV HRB-SS全长基因组cDNA克隆的重组质粒p FCV-HRB-SS。将重组质粒p FCV-HRB-SS转染猫肾细胞(CRFK),48 h即可观察到典型细胞病变,将病变细胞的上清接种未感染的CRFK细胞进行传代培养,连续传5代,从感染的细胞上清中获得拯救病毒。采用FCV VP1蛋白MAb,经间接免疫荧光试验检测,结果显示拯救病毒与亲本病毒均可观察到特异性绿色荧光,而未感染病毒的对照组细胞无荧光信号;各组细胞负染色后经电镜观察均可见病毒粒子呈典型的杯状结构。提取拯救病毒和亲本病毒基因组RNA,反转录为c DNA作为模板,经RT-PCR扩增、Kpn I酶切鉴定及测序分析,结果显示,拯救病毒含C^(5724)G位点突变的分子标记,消除了Kpn I酶切位点,不同于亲本病毒。上述结果表明获得拯救病毒r FCV HRB-SS。采用蚀斑形成试验和绘制病毒的一步生长曲线进一步检测r FCV HRB-SS,结果显示,r FCV HRB-SS与亲本病毒具有一致的复制水平和体外生长能力。本研究利用真核启动子构建的FCV HRB-SS株全长感染性cDNA克隆系统可直接在细胞内转录,减少了体外操作流程,有效防止了操作污染,具有操作简便,拯救效率高的优势,为后续FCV基因功能的研究和新型疫苗的研发奠定了基础。To establish a reverse genetic platform for feline calicivirus(FCV),the full-length genome of FCV HRB-SS strain was chemically synthesized with hepatitis D virus ribozyme(HdvRz)sequence inserted at the 3'end.The synthesized virus genome was cloned into the vector pOK12-CMV to obtain the recombinant plasmid pFCV-HRB-SS,which was transfected into Crandell-Rees feline kidney(CRFK)cells for virus rescue.Typical cell cytopathic effect(CPE)was observed until 48 hours after transfection of the recombinant plasmid pFCV-HRB-SS into CRFK cells.The supernatant from the cytopathic cells was serially passaged for five generations in CRFK cells,and the rescued virus was obtained from the supernatant of the infected cells.An indirect immunofluorescence test detected the virus using an MAb against the FCV VP1 protein.The results showed that specific green fluorescence could be observed in both the rescued and parental viruses.In contrast,the cells in the control group without virus infection had no fluorescence signal.The cells in each group were negatively stained and observed under an electron microscope.Virus particles,which have a typical cup-shaped structure,can be observed.The rescued and parental viruses'genome RNA was extracted to perform RT-PCR amplification,Kpn I digestion,and sequence analysis.The results showed that the rescued virus carries a molecular marker with a C^(5724)G point mutation,which abolishes the Kpn I restriction site and is distinct from the parental virus.It was shown that the virus was successfully rescued and named rFCV HRB-SS.The plaque formation test and one-step growth curve showed that rFCV HRB-SS exhibited replication and growth ability similar to the parental strain.The present study utilized a eukaryotic promoter-based FCV HRB-SS strain full-length cDNA infectious clone system,which allows for direct intracellular transcription,reduces operational steps,effectively prevents operational contamination,and has the advantages of easy operation and high rescue efficiency.This system provides a f

关 键 词：猫杯状病毒 感染性cDNA克隆 反向遗传操作系统 

分 类 号：S852.65[农业科学—基础兽医学]